Haynes:ExtFluorProtein: Difference between revisions
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by Karmella Haynes, 2014<br><br> | by Karmella Haynes, 2014<br><br> | ||
Principle: Fluorescent proteins that are gently extracted from cell samples can be detected on a plat reader. This procedure is for determining the yield of fluorescent protein relative to background protein levels. | Principle: Fluorescent proteins that are gently extracted from cell samples can be detected on a plat reader. This procedure is for determining the yield of fluorescent protein relative to background protein levels. | ||
MATERIALS | MATERIALS | ||
* Black opaque, clear bottom, flat-bottom 96-well plate (e.g. Corning COSTAR 3603) | * Black opaque, clear bottom, flat-bottom 96-well plate (e.g. Corning COSTAR 3603) | ||
* Fresh or thawed protein samples on ice | |||
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'''Settings for the BioTek Synergy H1 Plate Reader''' | '''Protocol > Action > Read Settings for the BioTek Synergy H1 Plate Reader''' | ||
{| | {| {{table}} | ||
|- | |- | ||
| Fluorophore || | | Fluorophore || Step label || Detect. Method || Read Type || Read Speed || Wavelengths || Excitation || Emission || Optics Position || Gain | ||
|- | |- | ||
| RFP | | RFP or mCherry || RFP || Fluorescence || End Point || Normal || 1 || 580 || 610 || Bottom || 100 | ||
|- | |- | ||
| GFP || | | GFP || GFP || Fluorescence || End Point || Normal || 1 || ### || ### || Bottom || 100 | ||
|} | |} | ||
'''Part 2: Measure background protein levels and normalize the fluorescence (IMPORTANT)''' | '''Part 2: Measure background protein levels and normalize the fluorescence (IMPORTANT)''' | ||
# Follow the [http://openwetware.org/wiki/Haynes:Bradford Bradford Assay] procedure. | # Follow the [http://openwetware.org/wiki/Haynes:Bradford Bradford Assay] procedure.<br>'''Helpful tip''' - to conserve precious samples, you can use 5 μL from each well in Part 1 for the bradford assay, instead of using 5 μL from the original sample tubes. | ||
# Use the calculated protein level from the Bradford procedure to normalize the fluorescence values: (fluorescence signal in 100 uL) / (calculated protein μg/μL * 100 μL) = Fluorescence per μg total protein. | # Use the calculated protein level from the Bradford procedure to normalize the fluorescence values: (fluorescence signal in 100 uL) / (calculated protein μg/μL * 100 μL) = Fluorescence per μg total protein. | ||
# In your figure (poster, paper, thesis, etc.) report the Fluorescence per μg | # In your figure (poster, paper, thesis, etc.) report the Fluorescence per μg protein. | ||
</div> | </div> |
Latest revision as of 13:43, 15 September 2014
Fluorescent Protein Measurement
by Karmella Haynes, 2014
Principle: Fluorescent proteins that are gently extracted from cell samples can be detected on a plat reader. This procedure is for determining the yield of fluorescent protein relative to background protein levels.
MATERIALS
- Black opaque, clear bottom, flat-bottom 96-well plate (e.g. Corning COSTAR 3603)
- Fresh or thawed protein samples on ice
EQUIPMENT
- Biotek Synergy H1 Plate Reader (or similar)
PROCEDURE
Part 1: Fluorescent protein measurement
- Follow the protein extraction protocol [link]. The final volume will be ~500 μL for 5x106 cells or less. The samples should include a negative control: non-fluorescent cells.
- Transfer 100 μL of each sample onto a well int eh 96-well plate. Avoid making bubbles.
- Use the table below to set up a New Protocol on the plate reader. If you do not know how to use the plate reader, ask someone to help you to set up the machine.
Protocol > Action > Read Settings for the BioTek Synergy H1 Plate Reader
Fluorophore | Step label | Detect. Method | Read Type | Read Speed | Wavelengths | Excitation | Emission | Optics Position | Gain |
RFP or mCherry | RFP | Fluorescence | End Point | Normal | 1 | 580 | 610 | Bottom | 100 |
GFP | GFP | Fluorescence | End Point | Normal | 1 | ### | ### | Bottom | 100 |
Part 2: Measure background protein levels and normalize the fluorescence (IMPORTANT)
- Follow the Bradford Assay procedure.
Helpful tip - to conserve precious samples, you can use 5 μL from each well in Part 1 for the bradford assay, instead of using 5 μL from the original sample tubes. - Use the calculated protein level from the Bradford procedure to normalize the fluorescence values: (fluorescence signal in 100 uL) / (calculated protein μg/μL * 100 μL) = Fluorescence per μg total protein.
- In your figure (poster, paper, thesis, etc.) report the Fluorescence per μg protein.